GSM8046861: 5h-Input-2; Mus musculus; OTHER
150 μg total RNA per technical replicate was isolated from naïve and activated CD8+ T cells. Size selection was first performed using a MEGAclear transcription clean- up kit (AM1908, Ambion) to deplete small RNA (< 200 nt). Purified large RNA was then fragmented to ~150 nt with magnesium RNA fragmentation buffer (E6150S, New England Biolabs) at 94 °C for 4 minutes. 10 ng of fragmented RNA were transferred into a new nuclease-free tube and used as “input”. The remaining RNA fragments were incubated with 2 μg cap-m7G antibody (RN017M, MBL) in IPP buffer (100 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40, 1U/μl RNase inhibitor) for overnight at 4°C. This antibody-RNA mixture was subsequently incubated with protein A/G UltraLink Resin for 3 hours at 4°C. RNA fragments with m7G were extracted by Trizol reagent and isopropanol precipitation. 1 ng of the cap-m7G-immunoprecipitated RNA is used as “m7G-IP” and the remaining is subjected to “FTO (+)” or “FTO (-)” treatment. One-third of the “m7G-IP” RNA was used as “FTO (-)” samples. The remaining “m7G-IP” RNA was used for FTO demethylation treatment at 37 °C for 20 minutes, the demethylated RNA was purified by phenol-chloroform extraction and labeled as “FTO (+)” samples. Both the “FTO (+)” and “FTO (-)” samples were individually immunoprecipitated with the m6A antibody using the EpiMark N6-Methyladenosine Enrichment Kit following the manufacturer's protocols. All samples, including input, m7G-IP, FTO (+), and FTO (-) RNA samples, were subjected to library construction using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (634413, Takara) according to the manufacturer's protocol. Sequencing was performed on an Illumina NovaSeq 6000 with PE150.